Live vaccine constituting minor risk for humans

ABSTRACT

A salmonella live vaccine produced from at least one attenuated immunologic live vaccine strain, characterized in that the vaccine strain has an envelope marker which results in an increased sensitivity of the vaccine strain toward a specific therapeutically effective antibiotic and has at least one chromosomal antibiotic resistance mutation for the attenuation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 08/802,127filed Feb. 19, 1997, now U.S. Pat. No. 6,136,325, which is acontinuation-in-part of application Ser. No. 08/300,600, filed Sep. 2,1994, now abandoned. The contents of the above patent and applicationsare herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a special use of a live vaccine, to new livevaccines not having been used before, to a method of producing suchvaccines as well as to suitable vaccine strains, especially salmonella.

2. Description of the Related Art

The majority of salmonella-conditioned Gastroenteritis infectiosa ofhumans are caused by contaminated animal products. Especially chickenand chicken eggs, respectively, being infected with the at presentpredominantly occurring serovar Salmonella enteritidis have increasinglybeen causing infections, recently. Nevertheless, generally all foodstuffs are affected which originate from animals kept in mass-rearing.Here, normally many animals are kept in confined space, promoting thespread of infections among the animal stock.

The risk of a transmission of the infection from the infected animal tohumans can be reduced by customary veterinary medical measures for theinterruption of infection chains. Furthermore, thorough compliance withI kitchen hygiene regulations during the processing of contaminatedanimal products can prevent a transmission to humans. However,especially the latter regulations are not always being considered duringthe storage and processing of food. Therefore, it is imperative to ruleout the possibility of infected animals being processed right from thebeginning. This can be achieved e.g. through a vaccination of the animalstock against salmonella infections.

Suitable salmonella live vaccines have to comply with various differentconditions:

1. The virulence of the vaccine strains used in the production of thevaccines has to be adjusted in a way that guarantees a non-apparentinfection on the one hand, and a sufficient persistence of the vaccinestrains in the host tissue on the other hand, as a prerequisite for highimmunogenicity.

2. Furthermore, the stability of the vaccine strains used with respectto their virulence and their protective properties has to be widelyassured, I.e. it has to be assured that they do not mutate back into thevirulent wild strain.

3. To allow for the reduction of the probability of infections it shouldbe ascertained that the vaccine strains are not permanently beingexcreted alive and that they can only service for a short period of timein the environment, respectively.

The above-mentioned three conditions, which a live vaccine has to complywith, are to be discussed in detail in the following. As described in 1,the production of a suitable salmonella live vaccine is based on areduction of the virulence (attenuation) of the pathogenic salmonellaand simultaneous preservation of their antigen structures, and thus, theimmunogenic effect in the host. One possibility is e.g., to employdeletion mutants, e. g. pur or aro auxotrophic clones, as vaccinestrains. The attenuation level of these vaccine strains depends upon thelack of metabolites in vivo, which possibly impedes an accurateadaptation to the host to be immunized. In this respect, it is referredto the EP 0 263 528 in which stable asp mutants of Salmonellatyphimurium with different virulence reduction levels are described.Vaccine strains with attenuation levels adapted to each of the differenthost species can be produced by selecting suitable asp mutants.

A further possibility for an attenuation consists of the employment ofvaccine strains, the virulence reduction of which can be traced back toa metabolism drift mutation (called stwd mutation or marker in thefollowing). The term “metabolism drift” comprises all essential enzymesand functionally important cell compartments, respectively, having beenfunctionally altered by mutations, as e.g. ribosome proteins, gyrase,RNA polymerase, permease, wherein, as a result of these mutations,translation, DNA replication, DNA transcription or permeation are moreor less distinctly disturbed. Such stwd mutants, furthermore, show aresistance with respect to specific antibiotics and other substances(noxious substances). Stwd mutants can easily be obtained inlaboratories as chromosomal antibiotic resistance mutants. In thisrespect, from the EP 0 263 528 e.g. stwd mutants with a resistanceagainst nalidixic acid (Nal), streptomycin (Sm) or rifampicin (Rif) areknown. Especially if several stwd markers are incorporated into onevaccine strain (double or triple marker vaccine strain), de factounlimited possibilities are obtained for the production of a desiredattenuation level adapted to suit every specific host species.

With respect to the prior art “attenuation by means of stwd mutations”it is referred to the following publications: DD-WP 155 294; DD-WP 218834; DD-WP 235 828; DD-WP 253 182; DD-NP 253 184; DD-WP 281 118; DD-WP294 420; EP 0 263 528.

A further (mentioned above under 2) condition is that the attenuatedvaccine strains obtained by mutation do not mutate back into thevirulent wild strain. The required stability can, on the one hand, beachieved by only employing vaccine strains with which no reversions canbe detected in vitro or whose reversion ratios are <10⁷. A furtherpossibility is to employ vaccine strains comprising several mutationswhich independently reduce virulence. Here, the probability of a backmutation can almost be excluded.

The final condition, mentioned above under 3 in connection with the term“interruption of infection chains”, especially concerns the risks withrespect to a possible excretion and permanent survival of the vaccinestrains outside the vaccinated host. In this respect, it is desirable toreduce the excretion and the capability of survival of the vaccinestrains in the environment. To guarantee a sufficient immune response,on the other hand, the capability of temporary survival of the vaccinestrains in the host tissue after e. g. oral or parenteral applicationshould only be slightly impaired or not at all. Vaccine strain mutantscomplying with such requirements are known e.g. from the DD-WP 218 836,DD-WP 231 491, DDWP 253 182, DD-WP 253 183, DD-WP 253 184, and EP 0 263528. In the prior art it is suggested to optimize suitable vaccinestrains by employing so-called anti-epidemic markers for the reductionof excretion and the capability of survival in the environment. The termanti-epidemic marker characterizes outer envelope mutations in a broadersense, causing a functional variation of the permeability barrier in theouter membrane.

Vaccine strains can be provided with different anti-epidemic markersdepending upon the intended application form. The anti-epidemic markersknown at present are divided into three groups, depending on thealterations they cause in the outer membrane of the vaccine strain. Thefirst group comprises the so-called hst markers. The incorporation of anhst marker causes the vaccine strain to become highly sensitive towardsbile, anionic detergents, macrolide antibiotics and other noxioussubstances. Owing to the high sensitivity towards bile, there is areduced excretion with feces caused by the inactivation of the vaccinestrains already occurring in the intestinal lumen. If vaccine strainbacteria are excreted, they only have a shortened survival time in theenvironment, due to the lack of the permeability barrier in the outermembrane against tensides and macrolides and other noxious substances.Therefore, an infection can almost be excluded when using vaccinestrains including hst markers. When employing the usual doses ofvaccine, vaccine strains comprising hst markers can only be appliedparenterally, however. If applied orally, due to the high sensitivitytowards bile, the virulence is influenced to such an extent that asufficient immune response can only be achieved by employing extremelyhigh doses of vaccine. Therefore, the solution for an oral applicationwould be to provide vaccine strains including an anti-epidemic markerfrom one of the other two known groups. One group comprises theso-called rbt markers (reversion to bile tolerance). The rbt marker canbe obtained by mutation from the hst marker. It provides the vaccinestrain with an anti-epidemic potency just as the hst marker does.However, in contrast to the hst marker the vaccine strain comprising anrbt marker is tolerant towards bile, and can therefore be applied orallywithout a reduction of the virulence impairing the vaccination effect.The same stands for a further group, the so-called rtt marker (reversionto tenside tolerance). The rtt marker can be obtained by mutation fromthe. rbt marker. The vaccine strain comprising the rtt marker istolerant towards tensides and simultaneously possesses a sufficientanti-epidemic potency due to the remaining high sensitivity towardsmacrolides and other noxious substances. Also the rtt marker strain canbe applied orally without any problems.

Taking this information and these publications as a basis, live vaccinescan be produced which comply with all conditions required. Theadaptation to the respective host can be effected, e.g. in animal testseries.

A further problem remains to be solved. Even the compliance with allprecautions does not exclude the possibility of a person dealing e.g.with the vaccination of the animals or the production of the vaccines,coming into contact with the in fact attenuated but, nevertheless, stillpathogenic salmonella vaccine strains. Where healthy people areconcerned, there is hardly any risk of an infection. However, if theimmune system of people is weakened (e.g. by an HIV-infection), thecontact with such vaccine strains can result in a salmonella infection.

Therefore, the object of the invention is to provide a live vaccineagainst salmonella infections, starting from the known prior art, beingoptimally attenuated for the host to be immunized, providing it with animmunity for reducing the excretion of wild strains when applied orallyor parenterally, and constituting only a minor risk for people,especially those with a weakened immune system, or none at all. Afurther object of the invention is to provide a method for producingsalmonella live vaccines optimally suited to the respective host byemploying significantly less animal experiments than the conventionalmethods. Finally, the invention is to provide salmonella live vaccinestrains for live vaccines suitable for chickens and poultry in general.

This object is attained by a specific live vaccine, a specific methodfor the production of salmonella live vaccines, and live vaccinestrains.

The art described in this section is not intended to constitute anadmission that any patent, publication or other information referred toherein is “prior art” with respect to this invention, unlessspecifically designated as such. In addition, this section should not beconstrued to mean that a search has been made or that no other pertinentinformation as defined in 37 C.F.R. §1.56(a) exists.

SUMMARY OF THE INVENTION

The invention concerns the use of a live vaccine strain known as such(see e.g., EP 0 263 528) for the production of a specific live vaccine.The known salmonella live vaccine strains comprise an envelope marker aswell as an attenuation marker (e.g. auxotrophy marker or stwd marker)providing them with an anti-epidemic potency (reduced excretion by thehost and reduced survival rate in the environment, respectively). Theenvelope marker being employed there, furthermore, causes asensitization of the vaccine strains towards macrolide antibiotics. Thisantibiotic sensitization has, hitherto, merely been employed for theselection of suitable envelope mutants, i.e. in the production ofvaccine strains. According to the invention it has been recognized forthe first time that the macrolide sensitivity of the vaccine strainscomprising an envelope marker can also function as a safety mechanismwhen employing the vaccine strains produced in this manner. The vaccineis to be designed such that, in the case of it causing an infection ofanother host, an effective therapeutical treatment of the infected hostcan be carried out by means of macrolides. This aim is achievedrelatively easily by selecting only such vaccine strains (provided withan envelope marker) for the production of the vaccine whose propagationcan be controlled by the application of justifiable doses of macrolideantibiotics.

The known salmonella live vaccine strains used in a specific manner showa sensitivity towards macrolides, due to their envelope marker. Inaddition to envelope mutants having an increased sensitivity towardshydrophobic antibiotics (macrolides, e. g. erythromycin) also otherenvelope mutants are described (I. A. Hancock, R. E. W.: Ann. Rev.Microbial. 1984, 38, 237-264), occasionally having differentpermeabilities with respect to a sensitivity towards hydrophilic,hydrophobic and polycationic antibiotics. Such envelope markers havebeen insignificant in the production of bacterial live vaccines,hitherto.

The invention also concerns live vaccines which are produced byincluding at least one attenuated live vaccine strain having an envelopemarker providing the vaccine strain with an increased sensitivitytowards a specific therapeutically effective antibiotic with theexception of macrolides. The live vaccine strains being employed in theproduction of the salmonella live vaccine according to the invention,therefore, comprise an envelope marker generally providing them with ananti-epidemic potency (interruption of infection chains) and,optionally, a sensitivity towards macrolides, but, in any case, anincreased sensitivity towards another specific therapeutically effectiveantibiotic. The vaccine strains can be detected relatively easily byemploying the selected specific therapeutically effective antibiotic, sothat the production of a vaccine strain provided with a suitableenvelope marker does not create a great problem. It is understood thatwith respect to an optionally required possibility of treatment for anunwanted infection caused by the vaccine, preferably such antibioticsare selected which constitute good results with respect to thesalmonella serovars employed.

Furthermore, it is understood that the selected live vaccine strains arederived from predominant serovars, those of salmonella chosen forpractical application including Salmonella typhimurium and Salmonellaenteritidis, accession numbers DSM 9361 and DSM 8432.

As mentioned above, there are various possibilities for the attenuationof live vaccines. One possibility is to include an auxotrophy marker(e.g. asp˜) into the vaccine strain. Preferably, the vaccine strain isprovided with at least one chromosomal antibiotic resistance mutation.The term chromosomal antibiotic resistance substantially comprises theabove-mentioned stwd markers. It has been proven that by employingselected stwd markers and by a specific combination of several suchmarkers, respectively, the attenuation level of the vaccine strain canbe adapted optimally. Upon the selection of such chromosomal antibioticresistance mutations for the attenuation of vaccine strains, it has tobe secured that they do not exclude the increased sensitivity caused bythe envelope marker towards the therapeutically effective antibiotic.Therefore, during development of the vaccine strain it is sensible tofirstly determine which therapeutically effective antibiotic is to beemployed for the possibility of treatment of the vaccine strain.Dependent upon this, the vaccine strain and its chromosomal antibioticresistance mutation, respectively, are selected for the attenuation ofthe vaccine strain.

According to a further preferred embodiment of the invention, theenvelope marker is selected such that it provides the vaccine strain(besides an anti-epidemic potency and an optional macrolide sensitivity)with an increased sensitivity towards an antibiotic of the groupincluding quinolons, chloramphenicols or tetracyclines. Especiallypreferred is an envelope marker providing the vaccine strain with anincreased sensitivity towards the antibiotic ciprofloxacin, presentlythe most effective antibiotic against salmonella. During the productionof the latter vaccine strain, it is especially advantageous to provide ametabolism drift mutation, resulting in a streptomycin and/or rifampicinresistance, for the attenuation. These antibiotic resistances do notinterfere with a possible sensitivity of the vaccine strain towards theantibiotic ciprofloxacin.

Depending on the desired effective spectrum, the salmonella live vaccineaccording to the invention can be produced from one or several vaccinestrains of different serovars of the O-groups B (e.g. Salmonellatyphimurium), D (e. g., Salmonella enteritidis), C (e.g., Salmonellainfantis) and E (e.g., Salmonella anatum) as mono, bi, tri ortetra-vaccine.

A further problem, already mentioned above, is constituted in that, dueto different sensitivities, a specifically adapted live vaccine has tobe provided for every host. In this respect, it is referred to thealready traded Salmonella typhimurium vaccine “Zoosaloral Dessau” whichsufficiently immunizes calves after a single oral application, however,does not protect chickens against an I. p. toxic infection until threetimes oral vaccination (Linde, K. et al.: Vaccine 1990, 8, 278-282).Zoosaloral is a vaccine from Impfstoffwerk Dessau-Tornau GmbH and hastwo auxotrophy markers Pur⁻ his⁻. The double marker attenuation is basedupon the purine dependence (deficiency of purine available in the host)and an accidental co-mutation in the rfb locus adjacent to the histidinoperon for the S-type polysaccharide synthesis, which co-mutationresults in the leaky function. Zoosaloral is overattenuated for chicks.With respect to the sensitivity towards Salmonella typhimruium,dependent upon the host species, a hierarchy mice>calves>chickens can beconstituted. From this it follows that e.g., Salmonella typhimruiumvaccine strains for chicks and chickens have to have a lesserattenuation level (in comparison with mice) for the compensation of thelesser sensitivity. It can be assumed that the same correspondinglystands for other salmonella serovars.

It has been found that the generation times of salmonella live vaccinesgenerally correlate with their attenuation levels. This correlationbetween generation time and attenuation level especially occurs if thevaccine strains have been provided with a chromosomal resistancemutation for the attenuation.

The relation between generation time and attenuation level permits arelatively reliable selection of vaccine strains being suitable for aspecific host without prior animal experiments. In this respect, asuitable live vaccine for the immunization of chicks and chickensproduced of at least one attenuated vaccine strain has a generation timethereof being about 28 to 34 minutes. Vaccine strains with suchgeneration times have a lower attenuation level (in comparison with thevaccine strains of calves and mice), compensating the small sensitivityof chicks/chickens towards e.g., Salmonella typhimurium (S. tm) andother salmonella serovars.

As mentioned above, an effective salmonella vaccine is especiallyinteresting with respect to the host species chicks/chickens. However,it has also been applied with ducks and ducklings and should be usefulfor poultry in general since they all have the relatively short livesproblem that allows for the possibility of live virus in their meat. TheSalmonella typhimruium stwd mutants, having been deposited/publishedhitherto, have no direct relation to this host species. Vaccines areintroduced which can be optimally attenuated for chicks/chickens, orhave already been optimally attenuated. In this respect, the vaccinestrain S. tm Nal 2/Rif 9/Rtt is emphasized which is optimally attenuatedfor chicks/chickens. The same stands for the vaccine strain S. tm Nal2/Rif 9 which has not been deposited, yet, with respect to thisapplication, but still is to be commented on. The vaccine strains citedhave a generation time of about 32 minutes. From this fact the“attenuation equivalent generation time” for the selection of optimallyattenuated other strains of the same or other serovars has been derived.Nevertheless, with respect to the selection of suitable vaccine strainsfor chicks/ chickens generation times vary from 28 to 34 minutes. Thisdiversity acknowledges the fact that chicks/chickens have differentsensitivities towards different strains and strain-dependent differencesin virulence is of each serovar, respectively. By means of a few testseries, from the preselected vaccine strains having generation timesbetween 28 and 34 minutes those can be chosen which are optimallyattenuated for chicks/chickens. Advantageously, this at least allows forthe animal experiments required in the preselection to be dropped.

Furthermore, with respect to the possible host chicks/chicken it isespecially interesting if, as proposed according to the invention, asalmonella live vaccine is used comprising a vaccine strain with anincreased sensitivity towards a specific therapeutically effectiveantibiotic. Chickens have a relatively short life span in comparisonwith other host species, as e.g., humans, calves or piglets. In general,they are slaughtered after a comparatively short rearing period and soldas frozen or fresh meat. Since the vaccination has not taken place veryfar in the past, it can be assumed that there are still live salmonellavaccine strain bacteria within the chickens at the time of slaughter.These can then find their way into the environment. For the normalpopulation the small amounts of germs in question are insignificant. Therisk of an infection can almost be excluded in this respect.Nevertheless, in individual cases risk patients with weakened immunesystems (e.g., people with the HIV-virus) can be infected and react withclinical symptoms. Especially with respect to these people, it isrequired that an infection caused by the vaccine strain may becontrolled rapidly and without any problems. With respect to this, theincorporation of a sensitivity towards therapeutically effectiveantibiotics as a safety and therapy marker is a sensible alternative,excluding all theoretical reservations.

The prototype of such a safety and therapy marker is the so-called ssqmarker. Vaccine strains having an ssq marker possess a hypersensitivitytowards quinolons, especially towards ciprofloxacin, at present, beingthe most effective antibiotic against salmonella. Also the ssq marker,like the above-mentioned hst, rbt and rtt mutations, is an envelopemutant and, therefore, also has a more or less distinctive anti-epidemicpotency dependent upon its bile and anionic detergent tolerance andsensitivity, respectively.

The invention not only deals with the production of especially safe livevaccines. A further object is to provide a method for the production ofa live vaccine optimally attenuated for a specific host employing hardlyany animal experiments.

The principle of this method is based on the fact that an attenuation ofvaccine strains (especially with respect to the incorporation of stwdmarkers) leads to a prolongation of the generation times in comparisonwith the wild strain. It has already been mentioned above that theprolonged generation times may allow for a conclusion with respect tothe attenuation level of the vaccine strains. In conformity with themethod according to the invention, it is therefore sufficient todetermine the specific prolonged generation time of a vaccine strainsuitable for a host in a single test (e.g., an animal experiment). Theprolonged generation time determined such can then serve as anapproximate figure for the following selection of further vaccinestrains (of the same serotype), or it can be transferred to otherserotypes of the same genus as an attenuation equivalent.

For the production of a vaccine optimally suited to chicks/chickens,e.g., a vaccine strain is selected, the generation time thereof beingprolonged to about 28 to 34 minutes in comparison with the wild strain(22 minutes). The vaccine strain employed may e.g., be attained from awild strain having been provided with a streptomycin (Sm) or nalidixicacid (Nal) marker. Through this, a prolongation of the generation timefrom 22 minutes to 25 to 29 minutes is achieved. Subsequently, arifampicin (Rif) marker is incorporated as a further marker prolongingthe generation time.

According to the essential features of the method a vaccine can beproduced:

in a first step, by isolating stwd mutants of a phenotype having aspecific antibiotic resistance and showing a generation time beingprolonged by about 3 to 6 minutes in comparison with salmonella wildstrains, for the purpose of producing strains with small or moderateattenuation,

in a second step, by again isolating stwd mutants of another phenotypewith an antibiotic resistance from these lesser or moderately attenuatedsingle marker strains, again having a generation time being prolonged byan additional 3 to 6 minutes, whereby a set of vaccine strain candidatesis being achieved having prolonged generation times graded from about 28to 34 minutes (wild strain about 22 minutes)—which with respect to S. tmin the mouse model (since the logarithm of an LD₅₀ correlates linearlywith the (prolonged) generation time) corresponds to graded LD₅₀ valuesof about 10⁵ to 10⁷ (wild strain about 10¹) cfu—then once orallyimmunizing ≧36 hours old chicks with 10⁹ cfu of the individual vaccinestrains of this set, after two weeks orally infecting the immunizedanimals with 10⁶ cfu of the wild strain, and finally favoring the S. tmNal 2/Rif 9 strain with a (prolonged) generation time of about 32minutes (and an i.p. LD₅₀ mouse of about 10⁶ cfu) as the favoriteprototype vaccine strain with respect to the relation of “maximumpossible prolongation of generation time/attenuation level and yetoptimal reduction of the excretion of wild strain”, for thedetermination of the “attenuation equivalent (prolonged) generationtime” for other strains of the same serovar and serovars with onlymoderate or lacking virulence towards mice, respectively.

in a third step, by incorporating an “ssq safety and therapy” markerinto the double marker vaccine strains attenuated by means of stwdmutation, for the optimization of the vaccine strains/increase of theacceptance, the safety and therapy marker approximately quadrupling thesensitivity towards ciprofloxacin (chloramphenicol, doxycycline, etc.)and simultaneously insignificantly reducing the excretion and capabilityof survival in the environment.

The sequence described is arbitrary, and noxious substance resistancephenotypes (DD-WP 235 828) may also be used.

Furthermore, the invention relates to specific salmonella vaccinestrains, having generation times of 28 to 32 minutes, as well as tovariants on these vaccine strains having higher or lower attenuationlevels and generation times between 28 and 34 minutes. Finally, the useof such vaccine strains for the oral immunization of chicks as well asthe oral and parenteral immunization of chickens against salmonellainfections is described.

The vaccine strains (with or without ssq markers) referred to in theclaims and the following examples (as well as having been deposited) areexamples for all the vaccine strain candidates with graded attenuationlevels which may be produced. They are suitable for the production ofimmunogenic live vaccines, especially for chicks/chickens according topropagation methods known as such. With respect to the specificallystated vaccine strains having ssq markers, (and deposit members DSM8433, 8435, 9362, 8434, 8441, 8432), generation times of about 32minutes are indicated. Further specific strains having ssq markers havegeneration times of about 28 minutes (deposit number 9361) and about 30minutes (deposit number 9360). This statement with respect to generationtime is not to be understood as a restriction. Taking strain-dependentdifferences in virulence (invasive capacity/colonization activity) intoaccount, generation times of 28 to 34 minutes are also possible asattenuation equivalents. All deposit numbers referred to herein are withDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, a BudapestTreaty Depository.

The live vaccine based on Salmonella typhimruium Nal 2/Rif9/Rtt DSM 8432has bene found to impede intestinal colonization by other bacteria orpathogens and thereby also protects the host against other infections.This effect is also predicted with the other strains of the invention.

In the following table the salmonella vaccine strains favored for thedifferent serovars are listed.

Salmonella Vaccine strains Laboratory serovar clone number Depositnumber*) typhimurium Ssq/Sm 60/Rif 42 4242 DSM 8433 enteriditis Ssq/Sm24/Rif 12 4266 DSM 8435 Ssq/Sm 24/Rif 12k 4298 DSM 9362 Ssq/Sm 24/Rif12g 4297 DSM 9361 Ssq/Sm 24/Rif 3 4296 DSM 9360 infantis Ssq/Sm 153/Rif7 4289 DSM 8434 anatum Ssq/Sm 81/Rif 21 4279 DSM 8441 typhimurium Nal2/Rif 9/Rtt 4223 DSM 8432 *)the microorganisms were deposited at: DSM -Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, MascheroderWeg 1 B, D-38124 Braunschweig, Germany

In the following the invention is to be described in detail by severalexamples of embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of the invention is hereafter described withspecific reference being made to the drawings in which:

FIG. 1 is a Graph showing Frequency (% positive samples) and Duration(last positive result).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Material and Method

Strains Used

wild strains

S. typhimurium (S. tm) 415 (Metschnikov-Institute, Moscow), i.p. LD₅₀mouse ≧10¹ cfu, generation time≈22 min.

S. enteritidis (S. ent) 318 (Prof. Selbitz, University of Leipzig), i.p.LD₅₀ mouse—10⁵ cfu, generation time—22 min.

S. infantis (S. inf) (Dr. Beer, Veterinärunter-suchungsamt Chemnitz),generation time—22 min.

S. anatum (S. ana) (Dr. Beer, Veterinärunter-suchungamt Chemnitz)generation time˜22 min.

wild strains having a neutral nalidixic acid and streptomycin resistancefor the detection of the reduced excretion upon immunizedchicks/chickens.

S. tm Nal/Sm, generation time≈22.5 minutes

S. ent Nal/Sm, generation time≈22.5 minutes

S. inf Nal/Sm, generation time≈22.5 minutes

S. ana Nal/Sm, generation time≈22.5 minutes

wild strains as examples for other double or triple marker mutants withlower (or higher) attenuation/correspondingly less or more prolongedgeneration time:

S. tm Ssq/Sm 60/Rif 42, generation time≈31 min., laboratory no. 4242;deposit no. DSM 8433

S. ent Ssq/Sm 24/Rif 12, generation time≈32 min. laboratory no. 4266;deposit no. DSM 8435

S. ent Ssq/Sm 24/Rif 12k, generation time≈32 min. laboratory no. 4298;deposit no. DSM 9362

S. ent Ssq/Sm 24/Rif 12g, generation time≈28 min. laboratory no. 4297;deposit no. DSM 9361

S. ent Ssq/Sm 24/Rif 3, generation time≈30 min. laboratory no. 4296;deposit no. DSM 9360

S. inf Ssq/Sm 153/Rif 7, generation time≈31 min. laboratory no. 4289;deposit no. DSM 8434

S. ana Ssq/Sm 81/Rif 21, generation time≈32 min. laboratory no. 4279;deposit no. DSM 8441

S. tm Nal 2/Rif 9/Rtt, generation time≈32 min. laboratory no. 4223;deposit no. DSM 8432 as a prototype strain (with optimal attenuation forchicks/chickens) for the determination of the “attenuation equivalent(prolonged) generation time”.

Nutrient Media

Nutrient agar (SIFIN, Berlin-Weifβensee)

Tryptose phosphate broth (Difco, U.S.A.)

Antibiotics nalidixic acid (CHINOIN, Budapest) wild strains MHK 6.2ug/ml streptomycin (Jenapharm) wild strains MHK 6.2 ug/ml rifampicin(UBM, Bucarest) wild strains MHK 12.5 ug/ml ciprofloxacin (Bayer) wildstrains MHK 0.05 ug/ml chloramphenicol (Berlin-Chemie) 2.0 ug/mldoxycycline (Jenapharm) wild strains MHK 4.0 ug/ml erythromycin (Abbott)wild strains MHK 60.0 ug/ml

Test Animals and Conditions of Keeping

Chicks of hens laying brown eggs from different breeding stations werekept in cages by 5-10 animals and fed with turkey feed as well as waterad libitum.

Oral Immunization

Groups of 10 chicks each had a single dose of 10⁹ cfu, partly 10⁸ cfu,of the respective vaccine strain applied orally into their gullets,either 36 hours after hatching or (for the purpose of comparison anddetermination of the optimal immunization age) on the fourth day oftheir lives. The immunization under practical conditions is alsopossible via the drinking water (after water deprivation for 4 hours anamount of drinking water of 2 ml/chick is taken in within 3 hours).

Oral Infection

Two to four weeks after the oral immunization the chicks orally received10⁶ cfu (or 10⁷ cfu) of the respective neutrally marked homologous wildstrain by means of a pipette.

Detection of the Excretion

vaccine strain S. tm Ssq/Sm 60/Rif 42, S. ent Ssq/Sm 24/Rif 12, S. infSsq/Sm 153/Rif 7 and S. ana SsqJSm 81/Rif 21: nutrient media containing100 ug rifampicin and 200 ug streptomycin/ml;

vaccine strain S. tm Nal 2/Rif 9 with or without rtt marker: nutrientmedia containing 100 ug rifampicin and 12.5, ug nalidixic acid/ml;

neutral Nal/Sm marked wild strains: nutrient media containing 100 ugnalidixic acid and 200 ug streptomycin.

Per chick group and day of examination, five fresh stool samples eachwere suspended in 2 ml physiological sodium chloride solution and,additionally, dilutions from 10⁻¹ to 10⁻⁴ were produced.

quantitative determination: 0.1 ml of the original suspension and thedilutions, were applied onto nutrient agar with each 1% lactose andsaccharose, 0.015% bromthymol blue (determination of the number ofcoli/enterobacteria germs) by means of a spatula. Parallel to this, bothwere applied onto the same medium containing the respective antibiotic.As a quantitative measure for the excretion and the reduction ofsalmonella colonization occurring in immunized chicks in comparison withthe controls, the number of salmonella colonies vs. the number ofenterobacteria colonies was determined in thousandths.

qualitative determination: an antibiotic bouillon was added to theremaining original suspension. After incubation for 24 hours at 37° C.,the salmonella were transferred onto nutrient agar containing therespective antibiotic additives.

The confirmation of the grown salmonella was effected serologically,biochemically, and through the determination of the markers.

As a quantitative measure for the excretion and the reduction ofsalmonella colonization occurring in immunized chicks in comparison withthe controls, the number of salmonella colonies vs. the number ofenterobacteria colonies was determined in thousandths.

EXAMPLE 1

S. tm: Isolation of spontaneous chromosomal antibiotic resistance clonesas (Nal-twd single and) Nal/Ri-stwd double marker strains having gradedprolonged generation times between about 29 to 34 (wild strain about 22)minutes for the isolation of a prototype vaccine strain for thedetermination of the “attenuation equivalent (prolonged) generationtime” for strains of the same serovar and serovars having only moderate(S. ent) or lacking (S. inf and S. ana) virulence towards mice,respectively. 10⁹ (and 10¹⁰ ) cfu of the wild strain are transferredonto nutrient agar containing 100 ug (or in two steps of 50 ug andafterwards 400 ug) nalidixic acid/ml by means of a spatula and incubatedfor approximately two days at 37° C. The resistance clones aretransferred onto nutrient agar, controlled with respect to obtainedresistance, and the less or more reduced extinction is determined(Spekol with tube samples, Zeiss-Jena, wave length 650 no, starting germcount 10⁷ cfu, incubation in shaking water bath for 3 hours at 37° C.).The generation times of clones appearing to be suitable are measured bymeans of the Abbott MS-2 test system. As described above, on nutrientagar with 400 ug rifampicin/ml, resistance clones are attained fromclone Nal 2 having a generation time of about 28 minutes. The generationtimes of these resistance clones are determined, and the Nal 2/Rifclones having graded prolonged generation times between about 29 and 34minutes are used for the determination of the approximate figure(prolonged) “generation time as an attenuation equivalent” (see example3).

EXAMPLE 2

Detection of obtained colonization activity of the neutrally Nal/Smmarked S. tm, S. ent, S. inf and S. ana wild strains in 17 to 25 daysold chicks.

17 to 25 days old chicks orally received 10⁶ (or 10⁷) cfu of neutrallymarked wild strains by means of a pipette. During a period of 10 to 15days the quantitative salmonella colonization density was determined incomparison with the number of enterobacteria germs.

In the selected infection model (dose 10⁶ to 10⁷ cfu), after oralinfection, either the excretion of the neutrally marked wild strainswith high germ counts already occurs after 24 hours, or the salmonellagerm counts gradually only reach their highest figures in the stoolbetween the third and the sixth day (maximum figures at a 50 thousandthof the total enterobacteria flora). After eight to ten days, the numberof salmonella colonies drops to a 1.0 to 0.1 thousandth of theenterobacteria flora, and remains in this range for a longer period oftime.

This colonization dynamism demonstrates that the neutrally Nal/Sm markedwild strains are suitable for the determination of a reduced wild straincolonization in immunized chicks.

EXAMPLE 3

Determination of the S. tm Nal 2/Rif prototype vaccine strain having a(prolonged) generation time/attenuation level optimally suited tochicks/chickens by means of the relation of maximum possible prolongedgeneration time/attenuation level and yet optimal reduction of excretionof wild strain, for the purpose of the determination of the “attenuationequivalent (prolonged) generation time” for strains of the same serovarand of serovars having only moderate or lacking virulence towards mice,respectively.

Chicks at the age of ≦36 hours were immunized orally with 10⁹ cfu of thedifferent strains from the set of S. tm double marker strains havinggraded generation times between 29 and 34 minutes and, after two weeks,were infected orally with 10⁶ cfu of the wild strain. Parallel to this,control chicks of the same age were infected orally.

In comparison with the controls, the immunized chicks which received thevaccine strains having graded generation times between 29 and 34minutes, show a significantly reduced excretion of the wild strain(determined from the enterobacteria colonization density in thousandth),especially in the first five to ten days after the oral challenge. Thevariation (reducing with time) lies within the range of one to two tenthpowers. From the sixth to the tenth day after the oral challenge thesalmonella colonization densities in immunized chicks come into closeralignment with those of the controls (in the 0.1 thousandth range ofenterobacteria germ counts). In individual cases, however, immunizedchicks may show a reduced excretion in the range of one log step, evenafter the tenth day.

With respect to vaccine strains having generation times of ≧33 minutesthe excretion of wild strain is reduced significantly less, wherein thedifference to the controls generally does not exceed a tenth power. Onthe basis of the relation: “maximum possible prolonged generationtime/attenuation level and yet optimal reduction of excretion of wildstrain”, the S. tm Nal 2/Rif 9 having a generation time of about 32minutes (and an i.p. LD₅₀ mouse of about 10⁶ cfu) was determined as aprototype vaccine strain, and its (prolonged) generation time of about32 minutes was used as the approximate figure “attenuation equivalent”.

EXAMPLE 4

Recognition/detection of the Salmonella typhimruium clone S. tm Nal2/Rif 9 (I. p. LD₅₀ mouse about 10⁶ (wild strain about 10¹ ) cfu;generation time prolonged from 22 to 32 minutes as an attenuationequivalent), optimally attenuated for chicks/chickens, as the favorablevaccine strain over the equivalent protective effect against a toxicinfection conveyed by means of an i.p. and oral immunization; (withrespect to a corresponding transfer of these regularities to further“enteritidis” salmonella):

a. Preliminary test of the extremely differentiated sensitivity ofchicks/chickens and mice towards Salmonella typhimruium by determiningthe i.p. LD₅₀ rate of the S. tm wild strain and the S. tm Nal 2/Rif 9vaccine strain for chicks and mice.

For mice and two-day chicks the different i.p. LD₅₀ rates of the S. tmwild strain and the S. tm Nal 2/Rif 9 vaccine strain as well as,additionally, the i.p. LD₅₀ rate of the wild strain for 17 days oldchicks, are depicted in table 1.

TABLE 1 S. tm wild strain and S. tm Nal 2/Rif 9 vaccine strain:comparative i.p. LD₅₀ rates of mice and chicks 17-day chicken S. tm2-day chicken (cfu) (cfu) ICR mice (cfu) wild strain 10⁶ 10⁸ 10¹ Nal2/Rif 9 10⁷ n.t. 10⁶

The LD₅₀ rates for chicks and mice according to table 1 show the lessersensitivity of chicks towards S. tm, which has to be compensated by alesser attenuation level of the vaccine strains.

b. Recognition of the Salmonella typhimruium vaccine strain optimallysuited to chicks/chickens (with or without an rtt marker as an envelopemutation optimizing the vaccine strain) over the equivalent protectiveeffect against a toxic infection conveyed by means of an i.p. or oralimmunization.

The equivalent protective effects against an LD₇₅: toxic infection,effected two weeks later, which can be attained through a single i.p.immunization with the vaccine strain S. tm Nal 2/Rif 9 and the strainsS. tm Pur-(i.p. LD₅₀ mouse 10^(7.5) cfu) and Zoosaloral (i.p. LD₅₀10^(8.2) cfu) being overattenuated for chicks, on the second day afterhatching: see table 2;

oral immunization with S. tm Nal 2/Rif9, S. tm Nal 2/Rif 9/Rtt; as wellas the calf vaccine Zoosaloral being overattenuated for chicks, within≦36 hours or on the fourth day after hatching: see table 3;

served as the criterion “optimal attenuation”.**

TABLE 2 Attainable immunity against a toxic infection in the case of asingle i.p. immunization with 10⁶ cfu of mutants having differentattenuation levels on the second day of life; challenge with 3 × 10⁸ cfuof the wild strain on the 16th day of life (mean of 3 exp.) i.p.immunization: 10⁶ cfu S. tm vaccine strain/test strain i.p. challengemortality (%) Nal 2/Rif 9 25 Pur˜81 25 Zoosaloral 30 control 75

TABLE 3 Attainable immunity against a toxic infection in the case of asingle oral immunization with 10⁹ cfu of the vaccine strains andZoosaloral, being overattenuated for chicks; challenge with i.p. 3 × 10⁸cfu of the S. tm wild strain on the 16th day of life (mean of 4experiments) S. tm vaccine oral immunization on strain/test with 2-day4-day strain (cfu) mortality (%) Nal 2/ 10⁹ 23 35 Rif 9 10⁸ 27 n.t. Nal2/Rif 9/ 10⁹ 24 40 Rtt 10⁸ 26 n.t. Zoosaloral 10⁹ 42 60 10⁸ 47 65control 75

As shown in table 2, the single i.p. immunization with all vaccinestrains reduces the mortality of an unphysiological toxic infection fromabout 75% to ≦30%.

As shown in table 3, this reduction in mortality as opposed toZoosaloral and metabolism drift mutants having generation times of ≧33minutes and an i.p. LD₅₀≧10^(6.5) cfu—can also be obtained by a singleoral immunization with the vaccine strain S. tm 2/Rif 9 (with or withoutrtt marker), i.e., therefore, this vaccine strain is optimallyattenuated for chicks.

Furthermore, table 3 shows the:

overattenuation of the calf vaccine Zoosaloral

less protectively effective immunization against a toxic infection onthe fourth day of life. Presumably, this is for reasons of the highercolonization resistance on the fourth day of life, which shouldinfluence the translocation and penetration rates of the vaccinestrains.

EXAMPLE 5

S. tm, S. ent, S. inf and S. ana: Isolation of spontaneous antibioticresistance clones as (single and) double marker vaccine strains beingoptimally attenuated for chicks/chickens by means of the “attenuationequivalent (prolonged) generation time” (see also example 1).

10⁹ to 10¹⁰ cfu of the wild strain are transferred onto nutrient agarcontaining 400 ug streptomycin/ml by means of a spatula and incubatedfor approximately two days at 37° C. The resistance clones aretransferred onto nutrient agar, controlled with respect to obtainedresistance, in the preliminary test the less or more reduced extinctionin comparison with the wild strain is determined (Spekol with tubesamples, Zeiss-Jena, wave length 650 nm, starting germ count 10⁷ cfu,incubation in shaking water bath for 3 hours at 37° C.) (see example 1),and the generation times of clones appearing to be suitable are measuredby means of the Abbott MS-2 test system. As described above, in thesecond step, from clones having generation times being prolonged byabout 3 to 6 minutes rifampicin resistance clones (nutrient agarcontaining 400 ug rifampicin/ml) are obtained, the generation timesthereof are determined, and Sm/Rif clones having generation times ofabout 28 to 32 minutes are favored as double marker vaccine strains.(The use of the Sm-stwd attenuation instead of the Nal-stwd attenuationis preferable, since the ssq marker (see example 6) as an envelopemutation—e. g. in Sm/Rif vaccine strains (upon abstaining from anNal-stwd attenuation as a gyrase mutation)—also provides ahypersensitivity against the at present most effective antibioticciprofloxacin.)

EXAMPLE 6

Additional incorporation of an ssq (“safety and therapy”) marker,optimizing the vaccine strain and increasing its acceptance, into thewild and vaccine strains, respectively. A fresh culture is suspended inPBS and treated with 100 ug/ml N-methyl-N-nitro-N-nitrosoguanidine (MNG,ZIMET, Jena) up to a survival rate of 10%. Then, it is incubated for 2hours at 37° C. in nutrient bouillon containing 0.4 ug chloramphenicol,and treated in the conventional manner with 1000 IU penicillin/ml.Clones lacking growth are obtained by means of the stamping technique onnutrient agar containing 0.4 ug chloramphenicol (0.01 ug ciprofloxacin,1.0 ug doxycycline)/ml. As ssq (supersensitivity to quinolons) strainssuch clones are determined and used as safety and therapy markersoptimizing the vaccine strain and increasing its acceptance, which:

do not grow on nutrient agar containing 0.4 ug chloramphenicol, 0.01 ugciprofloxacin or 1.0 ug doxycycline (as well as, mostly, 30 ugerythromycin)/ml,

show a desirable reversion frequency of ≦10⁷.

The reversion frequency of the envelope mutation is better determined onnutrient agar containing 20 or 30 ug erythromycin/ml, since, upon thehigh germ counts, the rest growth shifts towards a two steps higherconcentration of ciprofloxacin, chloramphenicol and doxycycline.

Suitable as vaccine strains are clones having an ssq marker, which,through mutagen treatment, undergo no or only a minor generation timeprolongation/attenuation caused by co-mutation.

EXAMPLE 7

Recognition of the S. typhimurium, S. enteritidis, S. infantis and S.anatum vaccine strain being optimally attenuated for chicks/chickens bymeans of the detection of the obtained or residual invasive capacity ofthe metabolism drift (stwd) single marker mutants and double markervaccine strains for chicks.

Chicks at the age of ≦36 hours were infected orally with 10⁹ cfu of therespective wild strains, stwd single marker mutants and stwd-doublemarker mutants, respectively. After five to eight days the chicks werekilled, the liver extracted aseptically, was homogenized, transferredonto nutrient agar, and the remaining material was mixed with nutrientbouillon. The grown colonies and cultures, respectively, were controlledwith respect to O-group identity and marker.

With respect to S. typhimurium and S. enteritidis, after five days thegerm counts/gram liver are in the range of about 10³ cfu, after eightdays they are in the range of about 10² cfu. In contrast to that, withrespect to S. infantis and S. anatum, the germ counts are in the borderarea of quantitative determination at about 10¹ to ≦10² cfu, and aftereight days the detection often can only be obtained by means ofpropagation. These lower germ counts/gram liver, found with respect toS. infantis and S. anatum, apparently correlate with U. Methner'sobservation (doctoral thesis, University of Leipzig, Veterinary MedicalFaculty, 1991) that S. infantis is less invasive for chicks.

The generally obtained invasive capacity of the favored vaccine strains(and single marker mutants) which determined from the numbers ofcolonies incubated does not quite reach the figures of the homologouswild strains, apparently is essential for the vaccination success(Barrow, P. A. et al., Res. Microbiol., 1990, 141, pp. 851-853).

EXAMPLE 8

Determination of the frequency in percentages-and the duration ofexcretion of the S. tm Nal 2/Rif 9 vaccine strain (with or without rttmarker) after oral immunization of ≦36 hours old chicks, and of the S.tm Nal 2/Rif-9 after oral immunization of five days old chicks.

The frequency in percentages in the tested stool samples and the maximumdetectable duration of excretion of the vaccine strain with or withoutrtt marker, dependent upon the chicks' age upon oral immunization isshown in FIG. 1.

FIG. 1 shows the frequency (percentage of positive samples afterpropagation) and duration (last positive result, border of detectionabout 10 germs/gram stool) of excretion of the vaccine strains S. tm Nal2/Rif 9 without (—) and with ftt marker (▴—) after a single oralimmunization of ≦36 hours old chicks with 10⁹ cfu as well as with S. tmNal 2/Rif 9 (individual figures not shown, (—)) on the fourth day oflife. (Mean of four to five experiments)

The following facts are shown in FIG. 1:

upon immunization within ≦36 hours after hatching, the S. tm Nal 2/Rif 9is excreted over the 32 days tested. After incorporating the rtt marker,the vaccine strain can rarely be detected three weeks after theimmunization.

upon immunization on the fourth day, the S. tm Nal 2/Rif 9 (without rttmarker) can only be detected occasionally after the 18th day, apparentlycaused by the already existing colonization resistance caused byanaerobic bacteria.

the frequency in percentages of positive accumulation cultures isanalogous to this.

With respect to the oral immunization within ≦36 hours, the S. tm Nal2/Rif 9 can still be detected in about 90% of the samples, with respectto the vaccine strain having been optimized with the rtt marker, onlyabout 40% of the stool samples are still positive.

The colonization dynamism of the favored vaccine strains S. tm Ssq/Sm60/Rif 42, S. ent Ssq/Sm 24/Rif 12 g, S. inf Ssq/Sm 153/Rif 7 and S. anaSsq/Sm 81 /Rif 21 was determined in that, after oral application of 10⁹cfu within ≦36 hours after hatching, the portion of the respectivesalmonella vaccine strain in the enterobacteria flora of the stoolsamples over a duration of two weeks was tested, wherein the favoredvaccine strains (see. above) showed an excretion behavior comparable tothe vaccine strain S. tm Nal 2/Rif 9/Rtt.

EXAMPLE 9

Reduction of the quantitative excretion of homologous neutrally Nal/Smmarked wild strains in immunized chicks in comparison with controls.

The ≦36 hours old chicks which received a monovalent oral immunizationby the vaccine strains S. tm Nal 2/Rif 9/Rtt, S. tm Ssq/Sm 60/Rif 42, S.ent Ssq/Sm 24/Rif 12, S. ent Ssq/Sm 24/Rif12k, S. ent Ssq/Sm 24/Rif 12g,S. ent Ssq/Sm 24/Rif 3, S. inf Ssq/Sm 153/Rif 7 and S. ana Ssq/Sm 81/Rif21, respectively were infected with 10⁶ (partly 10⁷) cfu of therespective homologous wild strain on the 17th day of life, parallel tocontrols of the same age.

In comparison with the controls, the immunized chicks in compliance withthe results obtained with respect the S. tm prototype vaccine strain Nal2/Rif 9—show a significantly reduced excretion of the wild strain,especially in the first five to ten days after the oral challenge(determined from the enterobacteria colonization density in thousandth).The variation (reducing with time) lies in the range of up to two tenthpowers. From the sixth to the tenth day after the oral challenge, thesalmonella colonization densities in immunized chicks come into closeralignment with those of the controls (in the 0.1 thousandth range ofenterobacteria germ counts). In individual cases, however, immunizedchicks may show a reduced excretion in the range of one log step, evenafter the tenth day.

A complete elimination of the salmonella bacteria within the individualinfected animal can hardly be expected, since, apart from S. gallinarumpullorum, these pathogens apparently behave like a “normal flora” withinchicks. The highly reduced excretion with respect to immunizedchicks/chickens should, in the medium term, lead to a salmonella freechicken stock, in combination with hygiene measures.

EXAMPLE 10

Increased sensitivity towards detergents

The vaccine strains S. tm Ssq/Sm 60/Rif 42, S. ent Ssq/Sm 24/Rif 12, S.ent Ssq/Sm 24/Rif 12k, S. ent Ssq/Sm 24/ Rif 12g, S. ent Ssq/Sm 24/Rif3, S. inf Ssq/Sm 153/Rif 7, S. ana Ssq/Sm 81/Rif 21, S. tm Nal 2/Rif9/Rtt show in comparison to wild strains an increased sensitivitytowards anionic detergents, especially towards sodium dodecyl sulphate(SDS). So the vaccine strains do not show growth on appropriate nutrientmedia (approximately without proteins) at a concentration of 0.5 mgSDS/ml the wild strains grow at 5 mg SDS/ml.

Vaccine strains suspended in physiological solution of sodium chloridebecome lysed at room temperature within 30 minutes to about 90% whereaswild strains show only a lysis rate of about 10%.

It can be concluded from these observations that the vaccine strainshave an increased sensitivity resp. reduced capability of survival inthe environment especially if measures for hygienic cleaning are taken.

EXAMPLE 11

The separation of vaccine strains from wild strains with respect to:

S. tm Ssq/Sm 60/Rif 42, S. ent Ssq/Sm 24/Rif 12, S. ent Ssq/Sm 24/Rif12k, S. ent Ssq/Sm 24/Rif 12g, S. ent Ssq/Sm 24/Rif 3, S. inf Ssq/Sm153/Rif 7 and S. ana Ssq/Sm 81/Rif 21 is obtained by their resistanceagainst 100 ug rifampicin and 200 ug streptomycin/ml nutrient agar aswell as the missing growth on nutrient agar containing 0.4 ugchloramphenicol (or 0.01 ug ciprofloxacin, 1.0 ug doxycycline and 30 ugerythromycin, respectively)/ml.

S. tm Nal 2/Rif 9/Rtt is obtained by its resistance against 100 ugrifampicin and 12.5 ug nalidixic acid*/ml nutrient agar as well as themissing growth on nutrient agar containing 30 ug erythromycin/ml.

*(The reduced resistance of the rtt strain against nalidixic acid incomparison with the initial Nal 2/Rif 9 strain is a consequence of thertt mutation (incorporated later) leading to a varied permeability ofthe outer membrane.)

EXAMPLE 12

Mass culture, preparation and use of vaccine strains.

For the production of live vaccines (double marker vaccine strainswithout ssq marker or) triple marker vaccine strains with ssq marker areincubated in a suitable full medium as a liquid culture until the end ofthe logarithmic phase. The bacterial suspensions are mixed with aconventional stabilizer and lyophilized.

The vaccine strains obtained in this manner are usually applied to ≦36hours old chicks in a single dose of 10⁸ to 10⁹ cfu. Chickens usuallyreceive a single immunization/boostering of 10⁹ cfu orally, or about 10⁸cfu parenterally, prior to the laying period.

While this invention may be embodied in many different forms, there areshown in the drawings and described in detail herein specific preferredembodiments of the invention. The present disclosure is anexemplification of the principles of the invention and is not intendedto limit the invention to the particular embodiments illustrated.Although the invention has primarily been described with reference toSalmonella vaccines, the inventive methods should be applicable toproducing vaccines and methods of protecting against acquiring diseasefrom handling vaccine or ingesting vaccinated poultry when the vaccineis directed to Shigella, Listeria, Pasteurella, Campylobacter and E.coli, as well.

This completes the description of the preferred and alternateembodiments of the invention. Those skilled in the art may recognizeother equivalents to the specific embodiment described herein whichequivalents are intended to be encompassed by the claims attachedhereto.

What is claimed is:
 1. A method of reducing intestinal colonization by awild strain of Salmonella typhimruium in ≦36 hour-old chicks by oraladministration with a therapeutically effective amount of a liveattenuated mutant of S.typhimurium, Nal2/Rif9/Rtt.